This project focuses on characterizing: (1) pigment-related genes (and their functions) involved in melanocyte differentiation, (2) the biochemistry of melanins formed and their photoprotective properties, and (3) the biology of melanocyte specific differentiation antigens that serve as melanoma-specific targets. Our studies have identified, isolated and characterized several different specific melanogenic enzymes and structural melanosomal matrix proteins that regulate the quality and quantity of melanin pigment produced within melanocytes. Many of these normal differentiation proteins serve as immunologic targets for malignant melanoma, including tyrosinase, TRP1/Tyrp1/gp75, TRP2/Dct, silver/Pmel17/gp100 and MART1. These proteins are encoded by genes that are specifically expressed by mammalian melanocytes and mutations in a number of these genes have now been shown to be involved in several different human clinical pigmentary diseases. Since these melanocyte differentiation proteins are known to provide highly specific targets for humoral and cellular immune responses against malignant melanoma, our laboratory has initiated new projects to identify novel melanosomal targets, to characterize mechanisms involved in inherited human pigmentary disorders such as albinism, and to examine the photoprotective properties of melanins in UV-irradiated skin of different phototypes. Although expression of these genes is specific to pigment producing tissues, they are independently regulated following stimulation or inhibition of differentiation by various paracrine and autocrine factors, such as ultraviolet radiation, agouti signal protein and melanocyte stimulating hormone. The phenotypic and functional properties of the melanins produced by these regulatory catalytic controls differ dramatically, and effects on the functional and photoprotective properties of those melanins are being characterized. We have used differential display to identify a subset of genes involved in modulating pigmentation in response to agouti signal protein and a particularly interesting ubiquitous transcription factor (ITF2) has been identified that regulates expression of pigment genes in melanocytes. Our current research in this area is aimed at further characterizing those gene products and the nature of the regulatory mechanisms involved. Individuals with fair skin and red hair have dramatically higher rates of photocarcinogenesis than do individuals with darker phenotypes, and thus MC1R receptor mediated signaling is important, not only to the regulation of skin and hair color, but to the photoprotection of those tissues. In a collaboration with the FDA, we have examined the photoprotection afforded by different types and amounts of melanins in human skin of varying phototypes. There is a surprisingly wide range of responses among individuals of the same racial/ethnic background and research is continuing into characterizing these responses. The processing and subcellular transport of tyrosinase (the critical rate-limiting enzyme in melanin biosynthesis) to melanosomes is compromised in all forms of albinism we have studied to date, showing that this disease is actually an ER retention disorder. We have established conditions to purify melanosomes at various stages of development and have begun identifying proteins specifically localized in those fractions. This study has provided critical information about the biogenesis and function of this organelle, and should hopefully identify new potential melanoma targets. In sum, our recent research has provided important advances in understanding the mammalian pigmentary system and how it is regulated in vivo at the biochemical and molecular levels. These studies have potential impact on the regulation of human pigmentation, improving photoprotection, and melanoma diagnosis and treatment.